1. Field of Invention
The present invention relates to a mass spectrometer in general and in particular to a mass spectrometer that employs one or more spatially non-linear fields to accelerate ions from an ion source to a detector.
2. Description of Related Art
Mass spectrometers are instruments that are used to determine the chemical composition of substances and the structures of molecules. In general they consist of an ion source where neutral molecules are ionized, a mass analyzer where ions are separated according to their mass/charge ratio, and a detector. Mass analyzers come in a variety of types, including magnetic field (B) instruments, combined electric and magnetic field or double-focusing instruments (EB or BE), quadrupole electric field (Q) instruments, and time-of-flight (TOF) instruments. In addition, two or more analyzers may be combined in a single instrument to produce tandem (MS/MS) mass spectrometers. These include triple analyzers (EBE), four sector mass spectrometers (EBEB or BEEB), triple quadrupoles (QqQ) and hybrids (such as the EBqQ).
In tandem mass spectrometers, the first mass analyzer is generally used to select a precursor ion from among the ions normally observed in a mass spectrum. Fragmentation is then induced in a region located between the mass analyzers, and the second mass analyzer is used to provide a mass spectrum of the product ions. Tandem mass spectrometers may be utilized for ion structure studies by establishing the relationship between a series of molecular and fragment precursor ions and their products.
Alternatively, they are now commonly used to determine the structures of biological molecules in complex mixtures that are not completely fractionated by chromatographic methods. These may include mixtures of (for example) peptides, glycopeptides or glycolipids. In the case of peptides, fragmentation produces information on the amino acid sequence.
One type of mass spectrometer is time-of-flight (TOF) mass spectrometers. The simplest version of a time-of-flight mass spectrometer, illustrated in FIG. 1A (Cotter, Robert J., Time-of-Flight Mass Spectrometry: Instrumentation and Applications in Biological Research, American Chemical Society, Washington, D.C., 1997), the entire contents of which is hereby incorporated by reference, consists of a short source region 10, a longer field-free drift region 12 and a detector 14. Ions are formed and accelerated to their final kinetic energies in the short source region 10 by an electric field defined by voltages on a backing plate 16 and drawout grid 18. The longer field-free drift region 12 is bounded by drawout grid 18 and an exit grid 20.
In the most common configuration, the drawout grid 18 and exit grid 20 (and therefore the entire drift length) are at ground potential, the voltage on the backing plate 16 is V, and the ions are accelerated in the source region to an energy: mv2/2=z eV, where m is the mass of the ion, v is its velocity, z the number of charges, and e is the charge on an electron. The ions then pass through the drift region 12 and their (approximate) flight time(s) is given by the formula:t=[(m/z)/2 eV]1/2D   (I).which shows a square root dependence upon mass. Typically, the length s of source region 10 is of the order of 0.5 cm, while drift lengths (D) ranges from 15 cm to 8 meters. Accelerating voltages (V) can range from a few hundred volts to 30 kV, and flight time are of the order of 5 to 100 microseconds. Generally, the accelerating voltage is selected to be relatively high in order to minimize the effects on mass resolution arising from initial kinetic energies and to enable the detection of large ions. For example, the accelerating voltage of 20 KV (as illustrated, for example, in FIG. 1) has been found to be sufficient for detection of masses in excess of 300 kDaltons.
A profile of the acceleration potential in the source region 10 (shown in FIG. 1A) is shown in FIG. 1B. The potential in this embodiment decreases linearly from a maximum value at the backing plate 16 (shown in FIG. 1A) to zero at the drawout grid 18 (Shown in FIG. 1A).
In recent years, the development of an ionization technique for mass spectrometers known as matrix-assisted laser desorption ionization (MALDI) has generated considerable interest in the use of time-of-flight mass spectrometers and in improvement of their performance. MALDI is particularly effective in ionizing large molecules (e.g. peptides and proteins, carbohydrates, glycolipids, glycoproteins, and oligonucleotides (DNA)) as well as other polymers. The TOF mass spectrometer provides an advantage for MALDI analysis by simultaneously recording ions over a broad mass range, which is the so called multichannel advantage. In the MALDI method of ionization, biomolecules to be analyzed are recrystallized in a solid matrix (e.g., sinnipinic acid, 3-hydroxy picolinic acid, etc.) of a low mass chromophore that is strongly absorbing in the wavelength region of the pulsed laser used to initiate ionization. Following absorption of the laser radiation by the matrix, ionization of the analyte molecules occurs as a result of desorption and subsequent charge exchange processes. In TOF instruments, all ion optical elements and the detector are enclosed within a vacuum chamber to ensure that ions, once formed, reach the detector without collisions with the background gas.
One of the performance criteria for a MALDI-TOF mass spectrometer is the resolving power. The resolving power represents the extent to which ions of different m/z ratios can be distinguished from each other. Ideally, nearly infinite resolving power could be attained if all ions having the same m/z ratio would arrive at the detector simultaneously. However, because MALDI generated ions are formed with a range of initial energies and are extracted from the ion source over a range of starting positions, the ions acquire a range of kinetic energies over a range of times and the resolving power is consequently diminished. Therefore, in order to compensate for these variations in ion starting conditions and in order to attain sufficient resolving power, design features are incorporated in the Time-of-Flight spectrometer.
A number of techniques have been developed to improve the mass resolution of time-of-flight mass spectrometers. The first major improvement to resolving power incorporated two design features that improved both mass resolving power and overall mass range. One of the design features was the development of a two-field ion source (Wiley, W. C., McLaren, I. H., Rev. Sci. Instrumen. 1955, 26, 1150-1157; Wiley, W. C., Science, 1956, 124, 817-820; Wiley, W. C. U.S. Pat. No. 2,685,035). Earlier ion sources used a single electric field for ion extraction that imposed a tradeoff between energy and space focusing. FIG. 2A shows a graph of the voltage potential versus the length S0 between the ion source (backing plate) and the drawout grid or exit grid. The voltage potential decreases linearly to reach zero volt at the exit grid, illustrated in FIG. 2A by a dotted vertical line. The focus position lies at a distance of 2S0 from the exit grid. The focus position is indicated on FIG. 2A by a vertical line.
In order to maximize energy resolution, high electric field strength was used to accelerate the ions to their final velocity quickly. However, this required an axially short ion source geometry. In order to achieve a space focus condition the detector is placed also at a short distance (2S0) from the ion source. Hence, the time of flight was not long enough to achieve mass separation. The total time-of-flight could only be increased by either lowering the electric field strength, consequently leading to lowering of the energy resolution, or increasing the length of the flight path by moving the detector well beyond the focus region.
Since the dominant parameter limiting resolving power is the initial energy spread it is determined that lengthening the flight path is the appropriate solution to increasing total flight time to separate masses. Using a two-field ion source, as shown in FIG. 2B, the space focus region could be located farther than 2S0 from the ion source at a distance which is a function of the two accelerating field strengths. Thus, while the low amplitude first accelerating field slightly reduced the energy resolution, the ability to achieve both space focusing and an increase in the total flight time for all ions yielded an overall increase in resolving power.
Another early design provided additional focusing by introducing an adjustable time delay between ion formation and application of an acceleration field (Wiley, W. C., McLaren, I. H., Rev. Sci. Instrumen. 1955, 26, 1150-1157; Wiley, W. C., Science, 1956, 124, 817-820; Wiley, W. C. U.S. Pat. No. 2,685,035). During this time, ions move to new locations in the ion source due to their thermal energies and, upon extraction, acquire total kinetic energies dependent on these new location. This energy focusing method, known then as time-lag focusing and now known as pulsed or delayed extraction, essentially attempts to transform the energy distribution of the initial ion population into a spatial distribution, thus reducing the temporal effect of the energy distribution at the space focus position. The combined use of time-lag and space focusing yields a significant increase in resolving power. However, the optimal time lag is mass dependent, limiting the m/z range that could be simultaneously measured.
Another technique for improving the resolving power is the reflectron or ion mirror which provides mass-independent ion focusing (Karataev, V. I., Mamyrin, B. A., Shmikk, D. V. Sov. Phys. Tech. Phys. 1972, 16, 1177.; Mamyrin, B. A., Karataev, V. I.; Shmikk, D. V.; Zagulin, V. A. Sov. Phys. JETP 1973, 37, 45.; Mamyrin, B. A., Shmikk, D. V. Sov. Phys. JETP 1979, 49, 762.; Mamyrin, B. A., Karataev, V. I.; Shmikk, D. V. U.S. Pat. No. 4,072,862). An ion mirror in its basic form is shown in FIG. 3A. Ion mirror 30 comprises simply a series of electrostatic diaphragms 32 that provide a retarding electric field with enough potential to reflect ions. Ions with different kinetic energies penetrate the mirror to different depths before being turned around and repelled from the mirror.
While all ions leave the mirror having exactly the same magnitude of energy with which they entered, those ions possessing the greater energy travel farther into the mirror before being repelled and thus experience a time delay that compensates for their higher velocity in the field-free region. The ions are then focused at a second space-focus position SFP2 where they achieve a higher resolving power than the first space-focus position SFP1 due to the additional energy focusing. As shown in FIG. 3B, the original ion mirror design generates a single, linear electric field behind a field isolating mesh 33 and is capable of first-order focusing. A subsequent design incorporates two fields and is capable of first or second-order focusing.
Mass spectrometers using linear-field focusing devices such as the two-field ion source (shown in FIG. 2B) and the two-field ion mirror generate adequate resolving power for applications having a relatively small initial ion energy distribution. However, for applications having a relatively large initial ion energy distribution, the achievable resolving power is diminished. This is expected since the relationship between energy, velocity and time is fundamentally non-linear, and linear-field devices provide only an approximation of complete temporal focusing. Electrospray ionization (ESI) and MALDI, the two major ionization methods used in biological research, both generate ion populations having a relatively large energy distribution. One approach to compensate for this, used more commonly with ESI, overcomes the current energy focusing limitation by delivering externally-generated ions to the TOF mass analyzer in a direction orthogonal to the analysis axis. Thus, while the overall magnitude of initial ion energy is relatively large, the magnitude along the analysis axis is minimal. For MALDI-TOF, however, the ionization process occurs within the source along the analysis axis. A large initial ion energy distribution is thus inherent to the analysis, presenting a need for improved focusing methods.
The fundamentally non-linear relationship between time and energy in ion motion indicates that the ultimate attainable resolving power can only be achieved using non-linear fields, and the development of focusing methods using such fields is recently building momentum. Several ion mirror designs using a non-linear field have been developed (Glashchenko, V. P.; Semkin, N. D., Sysoev, A. A., Oleinikov, V. A., Tatur, V. Yu. Sov. Phys. Tech. Phys. 1985, 30, 540-541.; Mamyrin, B. A. Int. J. Mass Spectrom. Ion Processes, 1994, 131, 1-19.; Rockwood, A. L. Proc. 34th ASMS Conf. on Mass Spectrom. & Allied Topics, 1986, Cincinnati, Ohio, 173.), while other designs have been proposed and/or patented (Yoshida, Y. U.S. Pat. No. 4,625,112; Frey, R., Schlag, E. W., U.S. Pat. No. 4,731,532; Kutscher, R., Grix, R., Li, G., Wollnik, H., U.S. Pat. No. 5,017,780; Managadze, G. G., Shutyaev, I. Yu. In Laser Ionization Mass Spectrometry, Vertes, A., Gijbels, R., Adams, F., Eds., John Wiley & Sons: New York, 1993, 505-549. Flory, C. A., Taber, R. C., Yefchak, G. E. Int. J. Mass Spectrom. Ion Proc. 1996, 152, 177-184; Doroshenko, V. M., Cotter, R. J. J. Am. Soc. Mass Spectrom., 1999, 10, 992-999; Cotter, R. J., Doroshenko, V. M. U.S. Pat. No. 6,365,892). All of which are incorporated herein in their entirety by reference.
Each of these designs provides only minor improvement to the resolving power achieved using linear-field ion mirrors, and each is suitable to only a relatively narrow initial range of ion energies. Non-linear-field mirrors that focus a broad range of initial ion energies have also been developed using either an entirely gridless design to achieve a single non-linear field (Cornish, T. J., Cotter, R. J. Rapid Comm. Mass Spectrom., 1993, 7, 1037-1040), or a gridded design generating a combination of linear and non-linear fields (Beussman, D. J., Vlasak, P. R., McLane, R. D., Seeterlin, M. A.; Enke, C. G. Anal. Chem. 1995, 67(21), 3952-3957).
While non-linear fields are theoretically preferable to linear fields, one of the drawbacks to generating such fields in ion mirrors is the result of their inherent radial field-inhomogeneity. Linear fields generate an electric potential that is constant in all directions orthogonal to the electric field. Thus, an ion beam entering a linear-field ion mirror at a fixed angle will experience the same force regardless of the entry point. In contrast, an ion beam entering a non-linear field will experience a force that depends on the exact point of entry. An ion beam of finite diameter will thus experience a range of non-linear fields, which reduces the resultant resolving power and radially disperses the ion beam, diminishing the ion transmission. A non-linear design has been developed that exploits the radial dispersion using a single-electrode can-shaped “endcap” ion mirror (Cornish, T. J., Cotter, R. J. Anal. Chem. 1997, 69(22), 4615-4618; Cornish, T. J.; Cotter, R. J. U.S. Pat. No. 5,814,813). A more recent and somewhat more complicated design also uses a minimum number (2 to 3) of electrodes to achieve the desired non-linear field (Zhang, J., Enke, C. G. J. Am. Soc. Mass Spectrom., 2000, 11(9), 759-764; Zhang, J., Gardner, B. D., Enke, C. G. J. Am. Soc. Mass Spectrom., 2000, 11(9), 765-769; Zhang, J., Gardner, B. D., Enke, C. G., Patent Pending).
In contrast to the developments in non-linear ion mirror design, the use of non-linear fields in ion source design is less prevalent. Several designs have been developed, for the analysis of gas-phase ions, where a “quadratic” non-linear ion-accelerating field is generated (Crane, W. S., Mills, A. P. Rev. Sci. Instrum. 1985, 56, 1723.; Hulett, L. D., Donohue, D. L., Lewis, T. A. Rev. Sci. Instrum. 1991, 62, 2131-2137; Rockwood, A. L., Udseth, H. R., Gao, Q.: Smith, R. D. Proc. 42nd ASMS Conf. on Mass Spectrom. & Allied Topics, 1994, Chicago, Ill., 1038). A mass spectrometer based on one of these designs, for the analysis of orthogonally-injected gas-phase ions, is commercially available (LECO Corp., product literature on the Jaguar LC-TOF mass spectrometer).
A separate design incorporating both linear and non-linear fields has been reported (Gardner, B. D., Holland, J. F. J. Am. Soc. Mass Spectrom., 1999, 10(11), 1067-1073), also for the analysis of gas-phase ions. A gridless ion source, which consequently generates a non-linear field by default, is also commercially available on a MALDI-TOF instrument, although the design has not been described (Kratos Analytical AXIMA).